Radiation therapy is widely used to treat localized prostate tumors. However, cancer cells often develop resistance to radiation through unknown mechanisms and pose an intractable challenge. Radiation resistance is highly unpredictable, rendering the treatment less effective in many patients and frequently resulting in cancer recurrence. There is a dire need to uncover the molecular events that cause cells to become resistant in order to improve radiation therapy. In our in-depth investigations of radiation-resistant prostate cancer (RR-PCa), we found that mitochondrial heat shock protein 90 (mtHsp90) level and mitochondrial metabolism were aberrantly high when compared to radiosensitive PCa. mtHsp90 is a chaperone that maintains the stability of many diverse proteins, including those that are necessary for tumor survival and metabolism. We further demonstrated that decreasing mtHsp90 protein level significantly restored the sensitivity of RR-PCa cells to radiation. Hence, our overarching hypothesis is that mtHsp90 defines resistance of prostate cancer cells to radiation, a premise that will be put under stringent testing in this proposal. Reactive oxygen species (ROS) are known to reduce the level of mtHsp90 by interfering with its transcriptional and post-translational levels. We screened 768 FDA-approved drugs in search of a potent drug that could raise the level of ROS, but not be toxic to normal cells. We found Azithromycin (AZM), a macrolide antibiotic, to be the most effective drug that selectively increases mitochondrial ROS and reduces mtHsp90 protein level. We further demonstrated that AZM enhances the death of cancer cells with radiation treatment. Encouraged by robust results, we aim to advance our findings in this project, test our hypotheses, and develop a paradigm for adjuvant treatment that will ultimately enhance radiation therapy as a more effective procedure. The goals are: Aim 1, to determine the functional importance of mtHsp90 in RR-PCa cell survival and adaptive metabolisms, Aim 2, to determine mechanistically how ROS down-regulates mtHsp90 protein level and sensitizes RR-PCa, and Aim 3, to validate in preclinical models if AZM-generated ROS down-regulates mtHsp90 and enhances radiation treatment. The results will establish a novel link between mtHsp90 and RR-PCa. This study using state-of-the art metabolomics, imaging techniques, and model systems and has the potential to be translated into a clinical practice because AZM already has a good safety record. In the era of precision medicine, we are confident of the prospects of our closely-focused studies, which will push boundaries and make radiation therapy a better procedure, and our approach will set a precedent for many cancer treatments where radiation therapy is preferred.